NEW YORK (GenomeWeb) – An international team used a combination of PCR, targeted enrichment, and metagenomic sequencing to generate the oldest bacterial genome so far — a sequence representing a 5,300-year-old Helicobacter pylori strain from the gut of Ötzi the Iceman.
Because H. pylori has only been detected in human hosts — with genetic patterns that vary geographically alongside human populations — the researchers were able to place the ancient H. pylori genome alongside strains found in present-day human populations to get new clues to the ancient colonization of Europe.
Although H. pylori carriage has diminished somewhat in present-day human populations due to enhanced hygiene and efforts to eradicate it, the bug has a long history of human occupation and was once found in roughly half of individuals. Together with its propensity for recombination, such features have made H. pylori a useful marker for tracking human migration and population mixing patterns.
In this case, the team determined that Iceman's ancient H. pylori genome, described online today in Science, represents an early European strain of the bug that persists in Europe today but has since mixed extensively with H. pylori that appear to have been introduced by populations arriving in Europe after Ötzi's time.
"We see a pattern [of H. pylori] that was present before major migration events came to Europe," first author Frank Maixner, coordinator of the European Academy of Bozen/Bolzano's Institute for Mummies and the Iceman, told GenomeWeb.
"Nowadays, in modern European strains, this [H. pylori strain] is still visible," Maixner noted. "So there seem to be really important factors in the genome of the Iceman [H. pylori strain] that were later kept, even though there was quite a high degree of introgression."
A great deal of research has been done on Ötzi the Tyrolean man's ancient remains were unearthed by hikers high in the Italian Alps near the Austrian border in 1991. A genome sequence for Ötzi was first reported in 2012 and subsequent research suggested he shared genetic ties to early European farmers.
From other analyses, researchers have learned that the Iceman was probably murdered — an arrowhead pierced one of his major arteries and there are signs he suffered a blow to the head. He also appeared to have arthritis, rib fractures, and risk variants related to heart disease in his genome.
On the infectious disease side, past work suggested Ötzi had intestinal parasites that may have been common at the time. Metagenomic sequencing uncovered DNA sequences that appeared to stem from Borrelia burgdorferi, a bacteria that causes Lyme disease, though it is unclear that he suffered from symptoms of that disease.
Likewise, Maixner and colleagues cannot be sure whether Ötzi experienced ill effects from H. pylori, a pathogen that causes stomach ulcers and/or stomach cancer in a subset of carriers. Their stomach proteome analysis uncovered inflammatory proteins that may have been produced by immune cells in response to the bug, though they have yet to detect histological signs of infection.
The team initially used PCR to detect H. pylori DNA in the gastrointestinal tract of the Iceman, who was briefly defrosted so researchers could obtain a dozen samples from his stomach and intestinal contents, as well as mucosal tissue from the stomach, through an existing incision.
The pathogen appeared to be present in all of the samples tested, particularly in samples from Ötzi's stomach.
To rule out contamination by modern day H. pylori, while generating additional sequences for the Iceman's H. pylori strain, the researchers turned to metagenomic and targeted enrichment sequencing, both done with Illumina HiSeq instruments.
Consistent with the ancient nature of the pathogen, the resulting H. pylori sequences contained DNA damage patterns that resembled those in other ancient samples.
In addition, Maixner noted, the team was encouraged to find more H. pylori reads in Ötzi's stomach samples than in samples from the lower intestine — a pattern that fits with the location of H. pylori in modern infections.
Using bioinformatic approaches designed to remove sequences from human DNA and other microbes, the researchers teased more than 15,000 H. pylori reads out of their metagenomic sequence data.
Those sequences, as well as reads produced with Agilent in-solution hybridization capture, were aligned to an existing H. pylori reference genome at an average depth of nearly 19-fold coverage.
The ancient genome contained roughly 43,000 SNPs, along with deletions and insertions not found in the reference strain. The available data suggested Ötzi was infected with a single H. pylori strain containing sequences associated with virulence.
A look at housekeeping genes from the strain in the context of 1,600 global strains from a a multilocus sequencing typing database suggest Ötzi was infected with a form of H. pylori that resembled strains currently found in human populations from Central and South Asia.
The researchers saw similar results when they compared the Iceman's H. pylori sequence to previously sequenced complete genomes from 45 modern H. pylori isolates — the analysis uncovered H. pylori sequences still found in Europeans, despite placing the ancient strain closest to H. pylori genomes from India.
Present-day European populations typically carry H. pylori strains showing ancestry from the same strain as well as a strain that's been linked to African populations, the researchers explained, hinting that the strain present in Europe in Ötzi's day has since mixed with others as new human populations arrived in the region.
"This would lead us to believe that the population that the Iceman strain belonged to must have been the original population that inhabited the stomachs of Europeans 5,300 years ago," co-author Yoshan Moodley, an integrative biology and evolution researcher affiliated with the University of Veterinary Medicine Vienna and the University of Venda in South Africa, said during a telephone briefing with reporters this week.
"Furthermore, the Iceman's strain shows very little evidence of African admixture," Moodley noted, suggesting "the waves of migration that brought the African Helicobacter pylori into Europe had not occurred — or at least not occurred in earnest — by the time the Iceman was around."
Along with ongoing efforts to characterize other microbes the Iceman may have carried in his gut, the researchers are currently summarizing findings from a multi-omics analysis of plant and animal material present in his stomach when he died, Maixner explained, which includes efforts to understand how this food was prepared.
The team also hopes mummy samples from other parts of the world such as South America and Korean might yield additional ancient H. pylori sequences that can further flesh out human migrations in other parts of the world.